Expression Of Full-length Receptor Research Articles

Expression of a recombinant full-length LRP1B receptor in human non-small cell lung cancer cells confirms the postulated growth-suppressing function of this large LDL receptor family member.

Low-density lipoprotein (LDL) receptor-related protein 1B (LRP1B), a member of the LDL receptor family, is frequently inactivated in multiple malignancies including lung cancer. LRP1B is therefore considered as a putative tumor suppressor. Due to its large size (4599 amino acids), until now only minireceptors or receptor fragments have been successfully cloned. To assess the effect of LRP1B on the proliferation of non-small cell lung cancer cells, we constructed and expressed a transfection vector containing the 13.800 bp full-length murine Lrp1b cDNA using a PCR-based cloning strategy. Expression of LRP1B was analyzed by quantitative RT-PCR (qRT-PCR) using primers specific for human LRP1B or mouse Lrp1b. Effective expression of the full length receptor was demonstrated by the appearance of a single 600 kDa band on Western Blots of HEK 293 cells. Overexpression of Lrp1b in non-small cell lung cancer cells with low or absent endogenous LRP1B expression significantly reduced cellular proliferation compared to empty vector-transfected control cells. Conversely, in Calu-1 cells, which express higher endogenous levels of the receptor, siRNA-mediated LRP1B knockdown significantly enhanced cellular proliferation. Taken together, these findings demonstrate that, consistent with the postulated tumor suppressor function, overexpression of full-length Lrp1b leads to impaired cellular proliferation, while LRP1B knockdown has the opposite effect. The recombinant Lrp1b construct represents a valuable tool to unravel the largely unknown physiological role of LRP1B and its potential functions in cancer pathogenesis.

Soluble Corticotropin-Releasing Hormone Receptor 2α Splice Variant Is Efficiently Translated But Not Trafficked for Secretion

CRH directs the physiological and behavioral responses to stress. Its activity is mediated by CRH receptors (CRH-R) 1 and 2 and modulated by the CRH-binding protein. Aberrant regulation of this system has been associated with anxiety disorders and major depression, demonstrating the importance of understanding the regulation of CRH activity. An mRNA splice variant of CRH-R2alpha (sCRH-R2alpha) was recently identified that encodes the receptor's ligand-binding extracellular domain but terminates before the transmembrane domains. It was therefore predicted to serve as a secreted decoy receptor, mimicking the ability of CRH-binding protein to sequester free CRH. Although the splice variant contains a premature termination codon, predicting its degradation by nonsense-mediated RNA decay, cycloheximide experiments and polysome profiles demonstrated that sCRH-R2alpha mRNA escaped this regulation and was efficiently translated. However, the resulting protein was unable to serve as a decoy receptor because it failed to traffic for secretion because of an ineffective signal peptide and was ultimately subjected to proteosomal degradation. Several other truncated splice variants of G protein-coupled transmembrane receptors regulate the amount of full-length receptor expression through dimerization and misrouting; however, receptor binding assays and immunofluorescence of cells cotransfected with sCRH-R2alpha and CRH-R2alpha or CRH-R1 indicated that sCRH-R2alpha protein does not alter trafficking or binding of full-length CRH-R. Although sCRH-R2alpha protein does not appear to function as an intracellular or extracellular decoy receptor, the regulated unproductive splicing of CRH-R2alpha pre-mRNA to sCRH-R2alpha may selectively alter the cellular levels of full-length CRH-R2alpha mRNA and hence functional CRH-R2alpha receptor levels.

Expression and functional analysis of the anaplastic lymphoma kinase (ALK) gene in tumor cell lines.

The initial identification of the ALK gene, expressed as C-terminal part of the transforming fusion protein NPM-ALK in the t(2;5)(p23;q35) lymphoma-associated chromosomal translocation, revealed a novel receptor tyrosine kinase (RTK). In order to expand the knowledge on ALK expression in the human system, we examined a panel of human cell lines for ALK expression and found that transcription is completely repressed in cell lines of entodermal origin (0/21). Furthermore, full length receptor expression is absent in cell lines of the hematopoietic system with the exception of t(2;5)-associated anaplastic large cell lymphomas lines (ALCL), which are known to express chimeric NPM-ALK mRNA. Cell lines established from solid tumors of ectodermal origin, including melanoma and breast carcinoma, exhibited widespread mRNA expression of the ALK receptor at a broad range (53/64), an association which was found to be strongest in cell lines derived from neuroblastoma (6/6), glioblastoma (8/8) as well as in cell lines established from Ewing sarcoma (4/4) and retinoblastomas (2/2). Because of the reported involvement of neutrophin tyrosine kinase receptors in autocrine differentiation in neuroblastomas, we analyzed cell lines positive for full length or chimeric ALK protein for the presence of phoshotyrosine residues within the intracellular region of ALK. While the constitutive activation of chimeric NPM-ALK molecules could be shown, no evidence was found for induced or constitutively activated ALK receptors in neuroblastoma, melanoma or breast carcinoma cell lines. Although the receptor could be shown to be consistently expressed with exclusive specificity in tissues developed from the ectoderm, our results do not support any involvement of ALK in the stimulation of tumorigenic cell growth or differentiation so far, indicating that ALK expression is a physiologic rather than a pathologic phenomenon.